Leaving the European Union would be like escaping from jail, Boris Johnson claimed as he said a business leader who was suspended over his support for Brexit paid a "heavy price".
Downing Street has insisted it did not put pressure on the British Chambers of Commerce (BCC) to suspend its leader over comments suggesting the country could have a "brighter" future outside the EU.
Number 10 said it was "surprised" by the views expressed by John Longworth, the BCC's director general and acknowledged that it "regularly" speaks to business organisations.
But a spokesman stressed that "no pressure" was put on the BCC to suspend Mr Longworth.
Mr Johnson claimed the business leader had been crushed by the agents of Project Fear - the label used by Brexit campaigners to criticise the tactics used by David Cameron and his allies backing a Remain vote.
On BBC One's Andrew Marr Show Mr Johnson hit out at the approach of the Remain camp.
"This is like the jailer has accidentally left the door of the jail open and people can see the sunlit land beyond," he said.
"And everybody is suddenly wrangling about the terrors of the world outside. Actually it would be wonderful.
"It would be a huge weight lifted from British business."
He added: "It is very sad that someone like John Longworth, who shares my view, who has great experience of British business and industry, should have paid quite a heavy price, it seems, for sharing that optimistic view."
Mr Johnson continued: "He had been asked to step down for expressing what I think is a passionate, optimistic view of this country's chances."
The London mayor, whose decision to back a Brexit came after weeks of uncertainty about his stance and questions about his own political ambitions, insisted that he was "certainly not" expecting to be the next prime minister.
He insisted that Mr Cameron's position would be secure even if the country voted to leave the EU on June 23 amid speculation he could be ousted from Number 10.
"To the best of my knowledge there is not a single EU leader in the last 20 years who has had to step down as a result of a referendum, whether on Europe or not," he said. "I think the whole thing is a load of cobblers."
The fate of Mr Longworth prompted Downing Street to insist that the decision was entirely one for the BCC.
The Sunday Telegraph said a friend of Mr Longworth claimed Downing Street had "bullied" and been "putting pressure" on BCC board members to suspend their director-general.
Senior Tory backbencher David Davis demanded a clear statement by Number 10 "that the Government was not involved in pressurising the BCC into suspending Mr Longworth".
He added: "We need to know there was no contact between ministers and their officials before Friday's BCC board meeting that took the decision to suspend its chief.
"Downing Street has form in this respect. It has already admitted it made a 'mistake' in adding the name of General Sir Michael Rose, the former special forces commander, to a letter from former military leaders supporting Britain's continued membership of the EU."
A Number 10 spokesman said: "Given that 60% of BCC members say they want to stay in the EU, No10 was surprised to see the director general of the organisation come out for Brexit.
"We are clear no pressure was put on the BCC to suspend him. Of course No10 talks to business organisations regularly - but, to be clear: no pressure was applied. This decision is entirely a matter for the BCC."
Meanwhile, Justice Secretary Michael Gove attempted to undermine the Prime Minister's repeated claims that EU membership made the UK "stronger" and "safer".
The Justice Secretary told The Sunday Times: "I think overall our national security is strengthened if we are able to make the decisions that we need and the alliances that we believe in outside the current structures of the of the European Union."
He claimed EU judges had taken decisions against the UK's national interests by dictating "what our spies can do and whether we can be kept safe".
Mr Gove added: "Our security and sovereignty stand together. I believe that there are better opportunities to keep people safe if we are outside the European Union."